The long term objectives of this project are to describe, at the molecular level, the DNA repair pathways in the nucleus and the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Chlamydomonas offers an ideal combination of genetic and molecular approaches in achieving these objectives. The present project focuses on the light-dependent repair of ultraviolet-light induced DNA damage The working hypothesis of this project is that the nucleus and chloroplast have their own DNA photolyases under separate genetic control. The first specific aim is to complete the sequence analysis of a DNA photolyase gene that has been cloned from this organism. Identification of the 5' end of the mRNA and confirmation of the intron-exon boundaries will be done using PCR technology or the isolation and sequencing of a full length cDNA. The second specific aim is to search for and clone a second DNA photolyase gene from Chlamydomonas. The approach is to use the most highly conserved exon of the cloned DNA photolyase as a probe with the assumption that a second DNA photolyase gene will have conserved sequences. The third aim is to isolate another photoreactivation deficient mutant using a gene disruption technique. The last aim is to use the cloned DNA photolyase gene or genes as probes to investigate the factors that influence the expression of Chlamydomonas DNA photolyase genes. The results of this research will greatly increase our knowledge of DNA repair in the plant kingdom which is important with the increasing concern over the depleting ozone layer and the resulting increase in ultraviolet radiation reaching the earth. Since DNA repair enzymes appear to be highly conserved, this research should contribute to our understanding of DNA repair in other eukaryotes as well.